Techniques for isolating microorganisms currently in use in microbiology analysis laboratories, generally require the seeding or innoculating of the microorganism in or on a culture medium such that the development of the microorganism can take place under physical and chemical conditions which are rigorously predetermined and hence comparative from test to test. Generally the microorganism is seeded onto the surface of a solid culture medium constituted by nutritive elements contained in a gel, e.g. in the form of a layer in a petri dish which can be incubated. Each bacteria of the innoculum which survives, therefore, cends to grow in an isolated colony. Bacteria can be extracted from such colonies, such colonies can be counted or measured and determinations of colony growth and bacterial multiplication can be effected as a function of time. Therefore a number of known substances capable of forming, in an aqueous medium, gels which can solidify to form the matrix in or on which such colonies can develop. Such substances include synthetic macromolecular products, such as polyacrylamides and polycarboxyvinyl components, and mineral substances such as the silicates. However, these substances are not amenable to simple production of suitable gels because they are not reversible, i.e. they are not in the form of simple powders which can be converted into gels which can be melted and resolidified, or in the form of materials which can be converted into powders for ultimate conversion into gels as need arises. In the case of the synthetic macromolecular substances described it is generally necessary to add a polymerization agent to initiate the formation of the gel and this makes them incompatible with the need for simple powders which can be converted to gels only by the addition of water.
There are also a number of natural macromolecular substances capable of forming gels under the action of temperature or heat. Such is the case with gelatins and pectins which may be compatible with most components of a culture medium but which have only limited applications in bacteriology since with many microorganisms the gel is subject to attack by the microorganism, e.g. by bacteria.
As a practical matter at the present time, only the natural agar-agar has characteristics which permit practically universal use as a gel-forming agent for culture media in bacteriology. It has been to date the only product capable of giving reversible and stable gels at melting points (about 100.degree. C.) substantially greater than the gelification temperature (about 40.degree. C.). They are also characterized by a low tendency to attack by bacteria and low or no toxicity.
Naturally agar-agar or gelose is obtained by extraction of various marine algae of the Rhodophyceae family. It is a complex mixture of polysaccharides having a polygalactan skeleton formed by repetition of alternating units of (1.fwdarw.3)beta-D-galactose and (1.fwdarw.4)3,6-anhydro-alpha-L-galactose which can generically be referred to as agarose and in its commercial form is more or less substituted. Pure agarose does not occur in nature.
The result is a product which is approximately insoluble in water at 25.degree. C. but which can be dispersed in water as the temperature approaches 100.degree. C. to yield a colloidal solution whose viscosity varies as a function of the origin of the product and its concentration. At a concentration of at least 0.5% by weight, it forms, upon cooling, gels which have good stability and are reversible, i.e. they melt upon heating and rigidify upon cooling to a temperature below 40.degree. C. These gels are characterized by their dynamic gelification temperature (passage from a solution state to the gel state on cooling) and by their isothermic gelification temperature (i.e. the minimum temperature at which a solution of agar-agar is able to be maintained indefinitely in liquid form without forming the gel). These parameters also vary as a function of the origin of the agar-agar, of the molecular weight thereof, of the production history and of the concentration of agar-agar in the gel.
The proportion of solid culture media using agar-agar as the gel forming substances generally involves mixing the agar-agar with suitable nutritive substances and then, after addition of water, solubilizing the mixture while assuring sterility by autoclaving.
There are commercially available dry powders which can be mixed with the nutritive substances or which contain the nutritive substances and are simply to be mixed with water.
The latter powders, generally nonsterile, are marketed in lots of at least 500 grams and the typical method of preparing a culture medium from them involves the following steps:
weighing out the desired quantity of powder (for example 40 grams per liter of water);
dissolving with boiling the weighed out quantity of powder in the requisite quantity of water;
placing the solution in an autoclave at 120.degree. C. for at least 20 minutes; and
distributing the solution to a multiplicity of culture vessels, e.g. petri dishes, manually or mechanically via a machine or automatic apparatus designed for this purpose.
These operations generally require about an hour and this, of course, precludes rapid preparation of unit doses of the powder, e.g. for a single culture test. In practice, it is necessary to prepare at least 20 to 40 petri dishes at a time which creates other disadvantages, such as the occupation of valuable laboratory space and time.